Golgi ␣-mannosidase II cleaves two sugars sequentially in the same catalytic site Niket Shah*, Douglas A. Kuntz†, and David R. Rose*†‡ *Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada M5G 1L7; and †Ontario Cancer Institute, Princess Margaret Hospital, Toronto, ON, Canada M5G 1L7 Edited by Gregory A. Petsko, Brandeis University, Waltham, MA, and approved April 17, 2008 (received for review March 4, 2008) Golgi ␣-mannosidase II (GMII) is a key glycosyl hydrolase in the Here we present structures of dGMII bound to its natural N-linked glycosylation pathway. It catalyzes the removal of two substrate (GnMan5Gn2) and an oligosaccharide (Man5) lacking the different mannosyl linkages of GlcNAcMan5GlcNAc2, which is the key terminal N-acetylglucosamine residue that is required for the committed step in complex N-glycan synthesis. Inhibition of this reaction. The substrate was isolated and purified through the use of enzyme has shown promise in certain cancers in both laboratory and multiple enzymatic treatments and separation by chromatography. clinical settings. Here we present the high-resolution crystal structure Crystallization and complex formation made use of a catalytically of a nucleophile mutant of Drosophila melanogaster GMII (dGMII) inactive nucleophile mutant. Analysis of the substrate–enzyme bound to its natural oligosaccharide substrate and an oligosaccharide interactions provides compelling evidence of an evolutionarily precursor as well as the structure of the unliganded mutant. These conserved mode of substrate binding and catalysis. These results structures allow us to identify three sugar-binding subsites within the allow for the formulation of the complete catalytic process, which larger active site cleft. Our results allow for the formulation of the involves the sequential cleavage of two different linkages in the complete catalytic process of dGMII, which involves a specific order of same catalytic site by substrate rearrangement. They provide a bond cleavage, and a major substrate rearrangement in the active structural explanation for the requirement of the key N- site. This process is likely conserved for all GMII enzymes—but not in acetylglucosamine for the reaction to occur, despite its distance the structurally related lysosomal mannosidase—and will form the from the scissile bonds. Furthermore, the results strongly support a basis for the design of specific inhibitors against GMII. model in which the ␣1,6-linked mannose is cleaved before the ␣1,3-linked saccharide. Finally, the possibility of exploiting differ- cancer therapy ͉ enzyme mechanism ͉ N-glycosylation pathway ͉ ences between the lysosomal and Golgi variants of the enzyme for x-ray crystallography ͉ glycoside hydrolase the purposes of greater specificity in chemotherapy is explored. he N-linked glycosylation pathway is a complex yet ubiquitous Results and Discussion Ϫ Tphenomenon in eukaryotic cells (recently reviewed in ref. 1). Substrate Binding and Catalysis. The Fo Fc electron density map Eukaryotic N-linked glycosylation begins in the endoplasmic retic- shows continuous electron density in the active site at 1.4-Å ulum (ER) with the transfer of a preformed oligosaccharide from resolution and allows for the unambiguous placement of all atoms dolichol to an asparagine residue on a nascent polypeptide. This of the substrate oligosaccharide GnMan5Gn [Fig. 1B and support- oligosaccharide is sequentially modified by enzymes in the ER and ing information (SI) Table S1]. Hydrogen bonds and hydrophobic Golgi apparatus resulting in a final oligosaccharide structure. The interactions are presented in Fig. 1C and Table S2. modification steps involve trimming by glycosyl hydrolases and The GnMan5Gn oligosaccharide binds in a large groove on the extension by glycosyl transferases. surface of the enzyme. This groove contains the site of the Golgi ␣-mannosidase II (GMII) is a glycosyl hydrolase that nucleophile (Asp-204), acid/base catalyst (Asp-341), and zinc ion resides in the Golgi apparatus of eukaryotes and plays a key role in and represents a region of extremely high degree of amino acid the N-linked glycosylation of proteins (2, 3). GMII has a high conservation (see Fig. 5A and Fig. S1). When this structure is degree of sequence conservation among many eukaryotes. It has compared with the unliganded D204A dGMII refined to 1.3-Å been classified as a family 38 glycosyl hydrolase and catalyzes the resolution, it is clear that substrate binding does not result in any removal of both ␣-1,3-linked and ␣-1,6-linked mannoses from noticeable conformational change. GnMan5Gn2 to yield GnMan3Gn2 (Gn, N-acetylglucosamine), Interestingly, almost all of the protein–carbohydrate interactions which is the committed step of complex N-glycan synthesis (Fig. 1A) take place at three saccharide positions: M5, M4, and G3 (Fig. 1C (4). The reaction requires the presence of the terminal Gn added and Table S2). Additionally, the ligand B factors as determined by to the glycan by N-acetylglucosaminyltransferase I (GnT I) and is the crystallographic structure solution indicate that M5, M4, and hypothesized to proceed via a covalent glycosyl–enzyme interme- G3 are the most stably bound in the crystal structure (Fig. 1B). diate, preceded and followed by oxocarbenium ion transition states These three positions represent the saccharides most distal from the resulting in retention of the stereochemistry of the substrate. nascent protein. Substrate recognition by sensing the distal Previous work has determined that whereas GMII is able to cleave positions of the oligosaccharide is similar to what is seen with two chemically distinct mannosyl linkages, it does so within a single lectins (10). catalytic site. Swainsonine, a plant-derived indolizidine alkaloid, is a potent inhibitor of GMII, with a Ki of 40 nM against the Drosophila Author contributions: N.S., D.A.K., and D.R.R. designed research; N.S. and D.A.K. performed melanogaster enzyme (dGMII) (5–7). Clinical trials suggested that research; N.S. and D.A.K. analyzed data; and N.S., D.A.K., and D.R.R. wrote the paper. swainsonine has therapeutic value because it reduces metastasis The authors declare no conflict of interest. and improves clinical outcome in cancers of the colon, breast, and This article is a PNAS Direct Submission. skin (8, 9). Unfortunately, there are side effects resulting from Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, swainsonine treatment because it also inhibits a structurally related www.pdb.org (PDB ID codes 3CZN, 3CZS, and 3CVS). lysosomal mannosidase involved in catabolic processes. A greater ‡To whom correspondence should be addressed. E-mail: [email protected]. understanding of the GMII catalytic process will allow for the This article contains supporting information online at www.pnas.org/cgi/content/full/ development of highly specific inhibitors and thus, more efficacious 0802206105/DCSupplemental. chemotherapeutics. © 2008 by The National Academy of Sciences of the USA 9570–9575 ͉ PNAS ͉ July 15, 2008 ͉ vol. 105 ͉ no. 28 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0802206105 Downloaded by guest on September 23, 2021 Fig. 2. GMII–substrate. GnMan5Gn (yellow) is bound in the dGMII(D204A) active site (white). Hydrogen bonds are depicted as green dashed lines, and a single zinc ion is presented in purple and aids to coordinate M5 in the catalytic site. Anchor Site. The required N-acetylglucosamine (G3) is stably bound in a tight pocket 13 and 14 Å from M5 and M4, respectively. It forms strong stacking interactions with a GMII-conserved tyrosine resi- due (Tyr-267), its acetyl position is buried in a hydrophobic patch Fig. 1. Golgi ␣-mannosidase II–substrate interactions. (A) Golgi ␣-mannosi- formed by a conserved tryptophan (Trp-299) and proline (Pro- dase II catalyzes the cleavage of two mannosyl linkages, an ␣1,3-linkage 298), and it forms a hydrogen bond with a conserved histidine between M3 and M4 and an ␣1,6-linkage between M3 and M5, converting (His-273). The necessity of this nonhydrolyzed anchor is clear when Ϫ GnMan5Gn2 to GnMan3Gn2.(B) GnMan5Gn fitted to the Fo Fc electron considering the nature of the substrate. Oligosaccharide molecules density in the dGMII active site. The electron density is contoured to 3.0 ␴. The are highly flexible both about their glycosidic bonds and in the crystallographic temperature factors represent the average values for the conformation of their saccharide rings. The presence of a stabilizing carbon and oxygen atoms of the saccharide. (C) dGMII–GnMan5Gn interac- tions as determined by HBPLUS/LIGPLOT. anchor such as the G3 assists in binding and orienting the substrate for the hydrolysis reaction as well as increasing the local concentration of the linkages to be cleaved by GMII. The active site of dGMII is made up of three sugar-binding sites: The Fo Ϫ Fc electron density map from the dGMII(D204A)- the catalytic, holding, and anchor sites (Fig. 2). Man5 structure refined to 1.6-Å resolution shows clear electron density for the oligosaccharide (Fig. S2). The G3-lacking Man5 Catalytic Site. The ␣1,6-linked mannose (M5) is tightly bound in the oligosaccharide binding to dGMII also demonstrates the necessity catalytic site, forming many hydrogen bonds and stacking interac- of the anchor site in proper substrate orientation. The ␣1,6- and tions with highly conserved residues in that region of the active site. ␣1,3-linked sugars bind to the catalytic and holding sites, respec- Hydroxyls at positions 2, 3, and 4 hydrogen-bond to two conserved tively, in a manner nearly identical to that seen in GnMan5Gn. tyrosines (Tyr-269 and Tyr-727), a histidine (His-471), and an However, because of the lack of the anchor saccharide, the other aspartic acid (Asp-472). In addition, the hydrophobic face of the mannose positions are quite different, with the M1- and M2- saccharide ring forms a stacking interaction with Trp-95, and there containing oligosaccharide ‘‘tail’’ extending out of the active site is some hydrophobic contribution from Tyr-727 as well.